Benjamin Singer, M.D., Ph.D. - US grants

Project Summary This proposal describes a five-year career development program designed to lead the PI to a career as an independent clinician scientist studying the intersection of medical critical illness, neuroimmunology, and vascular biology. Research plan: Long term brain dysfunction, including cognitive and affective disorders, is common among the 1.3 million patients who survive critical illness every year in the United States. While the population impact of brain dysfunction after critical medical illness is similar to morbidity associated with stroke and is an area of growing scientific interest, few studies investigate the mechanism of these changes, and there is little scientific basis for the rational design of treatments. The applicant has found that in a mouse model of sepsis, systemic illness results in long lasting neuroinflammation with infiltration of inflammatory cells, changes in resident microglial gene expression, and blood brain barrier dysfunction. In this proposal, the applicant will investigate the role of an endogenous danger signal, the protein S100A8/A9, in sustaining neuroinflammation and blood brain barrier dysfunction in a mouse model of sepsis, and determine if blockade of S100A8/A9 signaling ameliorates these effects. He will study the role of S100A8/A9 both in an in vitro model system and in vivo using a combination of pharmacologic and genetic approaches. These questions will also be extended to post mortem studies of neuropathology and gene expression in the brains of patients with sepsis. Applicant: The applicant holds M.D. and Ph.D. degrees and has completed specialty training in Internal Medicine and Pulmonary and Critical Care Medicine. He has previous experience in neuroscience research and using mouse models to study normal brain physiology and disease. The career development plan includes a period of mentored research aimed at developing new knowledge in both immunology and vascular biology that will greatly enhance his existing training and allow him to develop as an independent investigator in studying an intrinsically interdisciplinary problem in translational neuroscience. The training will include learning research techniques and acquiring scientific knowledge in the laboratories of and through meetings with the mentors and key collaborators, as well as didactic training, seminars, lab meetings, topic focused working groups, and national meetings. The training plan includes didactic training in grant writing and responsible conduct of research. The research environment provides intellectual interaction with investigators from neuroscience, immunology and vascular biology, as well as basic and clinical/translational scientists. Technology for advanced imaging, gene expression, and immunophenotyping is available. These opportunities will allow the applicant to be guided in developing both powerful investigative techniques and the intellectual tools for an independent career in a clinically important but underserved field of study.

Project Summary This proposal describes a supplement to a career development award. The underlying career development program is designed to lead the PI to a career as an independent clinician scientist studying the intersection of medical critical illness, neuroimmunology, and vascular biology. The supplemental funds will support applying the findings of the underlying award to model mechanisms of cognitive decline associated with systemic illness in Alzheimer?s disease. The proposed pilot experiments will provide critical preliminary data for an R01 proposal examining the interaction of neuroinflammatory priming that results from amyloid deposition and the neuroinflammatory response to sepsis, thus supporting the applicant?s career development and transition to independence. Research plan: Long term brain dysfunction, including cognitive and affective disorders, is common among the 1.3 million patients who survive critical illness every year in the United States. Hospitalization is associated with increased risk of incident dementia among older adults, and patients with underlying Alzheimer?s disease neuropathology are particularly vulnerable to long-lasting cognitive effects of acute illness. The applicant has found that in a mouse model of sepsis, systemic illness results in long-lasting neuroinflammation including infiltration of inflammatory cells, priming of microglial reactivity, and astrocyte activation which accompany deficits in affective behavior. These changes are driven in part by an endogenous danger signal, the protein S100A8/A9, which signals via the receptor TLR4. In patients who die of sepsis, brain expression of neuroinflammatory markers is related both to underlying sepsis and to neurodegenerative disease. The central hypothesis of this proposal is that ?-amyloid deposition in the brain increases vulnerability to the effects of sepsis survival and acts synergistically with the insults of sepsis to amplify persistent neuroinflammation after sepsis. In this pilot study, this hypothesis will be tested by examining hippocampal-dependent learning and memory, molecular markers of inflammation, microglial priming, and leukocyte infiltration in both wild-type and ?-amyloid overexpressing mice, and testing the role of TLR4 signaling in these changes. Applicant: The applicant holds M.D. and Ph.D. degrees, has completed specialty training in Internal Medicine and Pulmonary and Critical Care Medicine, and is currently conducting mentored research. The pilot studies proposed here will allow the applicant to apply insights, models, and techniques developed during the period of mentored research to the study of Alzheimer?s disease. These studies will thus support the applicant?s transition to independence while also generating new insights into potentially reversible mechanisms of cognitive decline in patients suffering from neurodegenerative disease.

Abstract Long-term cognitive impairment affects more than 70% of sepsis survivors, but the underlying mechanisms remain unknown. Though widely hypothesized, evidence of blood-brain barrier (BBB) dysfunction in septic patients is limited by practical barriers to diagnostic studies in critically ill subjects. While BBB breakdown and cognitive impairment are seen in animal models of sepsis, the complexity of sepsis in vivo and differences between animal and human responses means that animal models cannot unambiguously identify the circulating factors that cause brain injury in human sepsis. Therefore, we propose to develop the µSiM-hNVU as an `on-chip' platform featuring a human iPSC-derived neurovascular unit (NVU; brain microvascular endothelial cells, pericytes and astrocytes). The `blood side' will allow the flow-based introduction of blood- borne cells and molecules with known or hypothesized roles in sepsis related brain injury, and the `brain side' will feature iPSC-derived microglial cells serving as a reporter of the brain inflammatory status. The human NVU will be built on a device platform ? the µSiM ? featuring ultrathin silicon nanomembranes that provide for unhindered solute exchange between `blood' and `brain' compartments and glass-like optical quality for live cell imaging and high-resolution microscopy. In the R61 phase, the device platform will be advanced for ease-of- use including `plug-and-play' modules for flow and barrier measurements (TEER, diffusion), and compatibility with a small-volume, digital-ELISA assay for secreted proteins. The µSiM-hNVU will be validated with functional assays of blood-brain barrier (BBB) function, protein expression studies, and transcriptional analysis. We will also build a iPSC NVU in which each cellular component of the NVU carries the ApoE4 allele. The expression of the ApoE4 lipoprotein drives BBB dysfunction by a known pathway and increases the risk of cognitive impairment in humans and animals experiencing brain inflammation. We will use the ApoE4-NVU as a `diseased BBB on a chip? which we hypothesize will show enhanced vulnerabilities to candidate mechanisms of brain injury identified by our team and others. Specifically, we will test the hypotheses that 1) pre-activated monocytes invade the brain and drive microglial activation; 2) the damage associated molecular pattern (DAMP) complex S100A8/A9 drive BBB breakdown to promote leukocyte infiltration and neuroinflammation; and 3) circulating factors that degrade endothelial glycocaylx (e.g., heparinase) or contribute to systemic inflammation (cell-free hemoglobin) promote CNS infiltration of leukocytes and subsequent neuroinflammation.